Simultaneous separation and detection of nine kynurenine pathway metabolites by reversed-phase liquid chromatography-mass spectrometry: Quantitation of inflammation in human cerebrospinal fluid and plasma

The kynurenine pathway (KP) generates eight tryptophan (TRP) metabolites collectively called kynurenines, which have gained enormous interest in clinical research. The importance of KP for different disease states calls for developing a low-cost and high-throughput chromatography-mass spectrometry method to evaluate the potential of different kynurenines. Simultaneous separation of TRP and its eight metabolites is challenging because they have substantial polarity differences (log P = −2.5 to +1.3). A low-cost, reversed-phase LC-MS/MS method based on polarity partitioning was established to simultaneously separate and quantitate all nine kynurenine pathway metabolites (KPMs) in a single run for the first time in the open literature. Based on stationary phase screening and ternary mobile phase optimization strategy, high polarity KPMs were retained while medium and low polarity KPMs were eluted in a shorter time. After method validation, we demonstrated the applicability of this LC/MS/MS method by quantitative measurement of all nine KPM in cerebrospinal fluid (CSF) and plasma among two groups of human subjects diagnosed with depression. Furthermore, we measured the differential KPMs in these two groups of low and high inflammation and correlated the results with CRP or TNF-α markers for depression. Our proposed LC-MS/MS provides a new metabolite assay that can be easily applied in various clinical applications to simultaneously quantify multiple biomarkers in KP dysfunction.